Infectious hepatitis C viruses of genotype 3A and 4A and uses thereof

ABSTRACT

The present invention relates to molecular approaches to the production of nucleic acid sequences, which comprises the genome of infectious hepatitis C virus. In particular, invention provides nucleic acid sequences which comprise the genomes of infectious hepatitis C viruses of either genotype 3a (strain S52) or genotype 4a (strain ED43). The invention therefore relates to the use of the nucleic acid sequences and polypeptides encoded by all or part of the sequences in the development of vaccines and diagnostic assays for HCV and in the development of screening assays for the identification of antiviral agents for HCV. The invention therefore also relates to the use of viral particles derived from laboratory animals infected with S52 and ED43 viruses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority to and is a U.S. National Phase Application of PCT International Application Number. PCT/DK2010/050236, filed on Sep. 16, 2010, designating the United States of America and published in the English language, which is an International Application of and claims the benefit of priority to Danish Patent Application No. PA 2009 70143, filed on Oct. 2, 2009. The disclosures of the above-referenced applications are hereby expressly incorporated by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to molecular approaches to the production of nucleic acid sequences, which comprises the genome of infectious hepatitis C virus. In particular, the invention provides nucleic acid sequences, which comprise the genomes of infectious hepatitis C viruses of either genotype 3a (strain S52) or genotype 4a (strain ED43). The invention therefore relates to the use of the nucleic acid sequences and polypeptides encoded by all or part of the sequences in the development of vaccines and diagnostic assays for HCV and in the development of screening assays for the identification of antiviral agents for HCV. The invention therefore also relates to the use of viral particles derived from laboratory animals infected with S52 and ED43 viruses.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) has a positive-sense single-strand RNA genome and is a member of the genus Hepacivirus within the Flaviviridae family of viruses (Rice, 1996). As for all positive-stranded RNA viruses, the genome of HCV functions as mRNA, from which all of the viral proteins necessary for propagation are translated.

The viral genome of HCV is approximately 9600 nucleotides (nts) in length and consists of a highly conserved 5′ untranslated region (UTR), a single long open reading frame (ORF) of approximately 9,000 nts and a complex 3′ UTR. The 5′ UTR contains an internal ribosomal entry site.

The 3′ UTR consists of a short variable region, a polypyrimidine tract of variable length and, at the 3′ end, a highly conserved region of approximately 100 nucleotides. The last 46 nucleotides of this conserved region were predicted to form a stable stem-loop structure thought to be critical for viral replication.

The ORF encodes a large polypeptide precursor that is cleaved into at least 10 proteins by host and viral proteinases. These proteins are the structural proteins Core, E1, E2; p7; and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, NS5B. The predicted envelope proteins contain several conserved N-linked glycosylation sites and cysteine residues. The NS3 gene encodes a serine protease and an RNA helicase and the NS5B gene encodes an RNA-dependent RNA polymerase.

A remarkable characteristic of HCV is its genetic heterogeneity, which is manifested throughout the genome. The most heterogeneous regions of the genome are found in the envelope genes, in particular the hypervariable region 1 (HVR1) at the N-terminus of E2. HCV circulates as a quasispecies of closely related genomes in an infected individual. Globally, seven major HCV genotypes (genotypes 1-6) and multiple subtypes (a, b, c, etc.) have been identified.

The nucleotide and deduced amino acid sequences among isolates within a quasispecies generally differ by 1-2%; those of different strains/isolates differ by 2-10%, whereas isolates of different subtypes and genotypes usually vary by >20% and >30%, respectively. Genotypes 1, 2 and 3 are found worldwide and constitute more than 90% of the HCV infections in North and South America, Europe, Russia, China, Japan and Australia. Throughout these regions genotype 1 accounts for the majority of HCV infections but genotypes 2 and 3 each account for significant percentage of infections.

More than 80% of individuals infected with HCV become chronically infected and these chronically infected individuals have a relatively high risk of developing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The only currently licensed therapy for chronic hepatitis C, interferon-alfa2 (IFN) in combination with ribavirin, induces a sustained viral response in less than 50-80% of treated patients depending on genotype. Consequently, HCV is currently the most common cause of end stage liver failure and the reason for about 30% of liver transplants performed in the U.S.

In addition, a number of recent studies suggested that the severity of liver disease and the outcome of therapy may be genotype-dependent. In particular, these studies suggested that infection with HCV genotype 1b and 3a were associated with more severe liver disease and that HCV genotype 1a and 1b might be associated with a poorer response to IFN therapy. As a result of the inability to develop a universally effective therapy against HCV infection, it is estimated that there are still more than 40,000 new infections yearly in the U.S. and about 3 million worldwide. Moreover, since there is no vaccine for HCV and as mentioned no effective treatment, HCV remains a serious public health problem.

Despite the intense interest in the development of vaccines and therapies for HCV, progress has been hindered by the absence of a useful cell culture system and the lack of convenient small animal models for laboratory study. For example, while replication of HCV in several cell lines has been reported, such observations have turned out not to be highly reproducible. And as described below only the JFH1 starin of HCV genotype 2a can grow in culture. The chimpanzee is the only HCV pathogenesis animal model. Consequently, HCV has been studied mainly by using clinical materials obtained from patients or experimentally infected chimpanzees, an animal model whose availability is very limited.

However, several researchers have recently reported the construction of infectious cDNA clones of HCV, the identification of which would permit a more effective search for susceptible cell lines and facilitate molecular analysis of the viral genes and their function.

Kolykhalov et al., (1997) and Yanagi et al. (1997, 1998) reported the derivation from HCV strains H77 (genotype 1a) and HC-J4 (genotype 1b) of cDNA clones of HCV that are infectious for chimpanzees. Subsequently, several other cDNA clones of genotype 1a (strains HCV-1 and TN), 1b (strains Cont and HCV-N) and 2a (strains J6 and JFH1) were developed. However, while these infectious clones will aid in studying HCV replication and pathogenesis and will provide an important tool for development of in vitro replication and propagation systems, it is important to have infectious clones of all major HCV genotypes, given the extensive genetic heterogeneity of HCV and the potential impact of such heterogeneity on the development of effective therapies and vaccines for HCV.

In addition, synthetic chimeric viruses can be used to map the functional regions of viruses with different phenotypes. In flaviviruses and pestiviruses, infectious chimeric viruses have been successfully engineered to express different functional units of related viruses and in some cases it has been possible to make chimeras between non-related or distantly related viruses. For instance, the IRES element of poliovirus or bovine viral diarrhea virus has been replaced with IRES sequences from HCV.

The construction of an infectious chimera of two closely related HCV subtypes has been reported. The chimera contained the complete ORF of a genotype 1b strain but had the 5′ and 3′ termini of a genotype 1a strain (Yanagi et al., 1998).

Recently, it was shown, that transfection of RNA transcripts from cDNA clone of genotype 2a isolate JFH1 into Huh7 hepatoma cells led to productive infection of these cells with JFH1 virus (Wakita 2005, Zhong 2005). It is not known, why JFH1 can grow in cell culture and other HCV isolates cannot. To exploit the exceptional growth characteristics of JFH1 in cell culture, the construction of JFH1-based intra- and intergenotypic recombinants became a research focus. Thus, intragenotypic and intergenotypic recombinants have been constructed containing non structural proteins NS3-NS5B of genotype 2a isolate JFH1 and Core, E1, E2, p7, and NS2 from genotype 1a (strain H77 and TN), 1b (strain J4 and Con-1), 2a (strain J6), 2b (strain J8), 3a (strain S52, DBN, and 452), 4a (strain ED43), 5a (strain SA13), 6a (strain HK6a), and 7a (strain QC69). Transfection of RNA transcripts of cDNA clones of these recombinants led to productive infection of Huh7.5 human hepatoma cells (Pietschmann 2006, Gottwein 2007, Scheel 2008, Jensen 2008, Gottwein, 2009). However, for most of the intergenotypic recombinants, viability in Huh7.5 cells required acquisition of cell culture adaptive mutations, possibly enabling interaction of proteins of different genotype isolates. J6/JFH1 has also been found to be viable in chimpanzees and in the SCID-uPA mouse model (Lindenbach 2005, Lindenbach 2006).

SUMMARY OF THE INVENTION

The present invention relates to nucleic acid sequences, which comprise the genomes of infectious hepatitis C viruses and in particular, nucleic acid sequences which comprises the genome of infectious hepatitis C viruses of genotypes 3a (strain S52) and 4a (strain ED43).

The present invention also relates to a method for producing a hepatitis C virus comprising transfecting a host cell with an RNA transcript of the nucleic acid of the present invention.

The invention further relates to polypeptides encoded by a nucleic acid sequence of the present invention.

An aspect of the present invention relates to a method for assaying candidate antiviral agents for activity against HCV, comprising; exposing a cell or a laboratory animal model containing the hepatitis C virus to the candidate antiviral agent; and measuring the presence or absence of hepatitis C virus replication or correlates thereof in the cell or in the animal.

The present invention also relates to an antiviral agent identified as having antiviral activity for HCV by the methods described herein.

In addition, the present invention relates to an antibody to the polypeptides and the hepatitis C viruses of the present invention.

The present invention relates to a composition comprising nucleic acid molecule and/or polypeptides of the present invention suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Phylogenetic tree of pS52, pED43 and representative HCV cDNA clones and isolates of HCV genotypes 1-4.

FIG. 2 Course of infection with HCV following intrahepatic transfection of chimpanzees 5276 (FIG. 2A) and 5300 (FIG. 2B) with RNA transcripts of pS52 (genotype 3a) and pED43 (genotype 4a).

FIG. 3 Genetic heterogeneity of S52 virus population from chimpanzee acute phase plasma pool.

FIG. 4 Comparison of ORF sequence of S52 with that of other genotype 3a isolates

FIG. 5 Genetic heterogeneity of ED43 virus population from chimpanzee acute phase plasma pool.

FIG. 6 Comparison of ORF sequence of ED43 derived from chimpanzee plasma pool with ORF of other genotype 4a isolates.

FIG. 7 3′ UTR Variable Region of pS 52 (nucleotides 9403 to 9448 of SEQ ID NO: 3) and other genotype 3a isolates.

FIG. 8 3′ UTR variable region of pED 43(nucleotides 9365to 9416 of SEQ ID NO: 4) and other genotype 4a isolates.

The present invention will now be described in more detail in the following.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic Acid Molecules and Sequence Identity

It is an object of the invention to provide nucleic acid sequences, which encode infectious hepatitis C viruses. Such nucleic acid sequences are referred to as “infectious nucleic acid sequence”, “nucleic acid sequences of the invention” or “nucleic acid molecules of the present invention” throughout the application.

For the purposes of this application, nucleic acid sequence refers to RNA, DNA, cDNA or any variant thereof capable of directing synthesis of a hepatitis C virus polypeptide by a suitable host organism. It is to be understood that nucleic acid sequences encompasses nucleic acid sequences, which due to degeneracy, encode the same polypeptide sequence as the nucleic acid sequences described herein.

As commonly defined “identity” is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. Thus, in the present context “sequence identity” is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.

To determine the percent identity of two nucleic acid sequences or of two amino acids, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions)×100). In one embodiment the two sequences are the same length.

One may manually align the sequences and count the number of identical nucleic acids or amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs. BLAST nucleotide searches may be performed with the NBLAST program, score=100 , word length=12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score=50 , word length=3 to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilised. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilising the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See the world wide web internet site “ncbi.nlm.gov”. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (world wide web internet site “ncbi.nlm.gov/cgi-bin/BLAST”). Generally, the default settings with respect to e.g. “scoring matrix” and “gap penalty” may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous.

The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

The present invention relates to nucleic acid sequence, which comprises the genome of an infectious hepatitis C virus of genotype 3a or 4a.

An aspect of the present invention relates to an isolated nucleic acid molecule which encodes human hepatitis C virus of genotype 3a, wherein said molecule encodes human hepatitis C virus of genotype 3a with the amino acid sequence according to that of SEQ ID NO: 1 or an amino acid sequence that has a sequence identity of at least 98% to that of SEQ ID NO: 1, such as 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 98.1% identity, 98.2% identity, 98.3% identity, 98.4% identity, 98.5% identity, 98.6% identity, 98.7% identity, 98.8% identity, 98.9% identity, 99% identity, 99.1% identity, 99.2% identity, 99.3% identity, 99.4% identity, 99.5% identity, 99.6% identity, 99.7% identity, 99.8% identity, or 99.9%.

Another aspect of the present invention relates to an isolated nucleic acid molecule which encodes human hepatitis C virus of genotype 4a, wherein said molecule encodes human hepatitis C virus of genotype 4a with the amino acid sequence according to that of SEQ ID NO: 2 or an amino acid sequence that has a sequence identity of at least 98% to that of SEQ ID NO: 2 such as 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 98.1% identity, 98.2% identity, 98.3% identity, 98.4% identity, 98.5% identity, 98.6% identity, 98.7% identity, 98.8% identity, 98.9% identity, 99% identity, 99.1% identity, 99.2% identity, 99.3% identity, 99.4% identity, 99.5% identity, 99.6% identity, 99.7% identity, 99.8% identity, 99.9 or 99.9%.

Another aspect of the present invention relates to a nucleic acid molecule that encodes human hepatitis C virus of genotype 3a comprising the nucleic acid sequence according to SEQ ID NO: 3.

In an embodiment of the present invention, the nucleic acid molecule encoding human hepatitis C virus of genotype 3a comprises the nucleic acid sequence according to SEQ ID NO: 3 or nucleic acid sequence with a sequence identity of at least 98% to SEQ ID NO: 3, such as 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 98.1% identity, 98.2% identity, 98.3% identity, 98.4% identity, 98.5% identity, 98.6% identity, 98.7% identity, 98.8% identity, 98.9% identity, 99% identity, 99.1% identity, 99.2% identity, 99.3% identity, 99.4% identity, 99.5% identity, 99.6% identity, 99.7% identity, 99.8% identity, 99.9 or 99.9%.

Another aspect of the present invention relates to a nucleic acid molecule that encodes human hepatitis C virus of genotype 4a comprising the nucleic acid sequence according to SEQ ID NO: 4.

In an embodiment of the present invention, the nucleic acid molecule encoding human hepatitis C virus of genotype 4a comprises the nucleic acid sequence according to SEQ ID NO: 4 or nucleic acid sequence with a sequence identity of at least 98% to SEQ ID NO: 4, such as 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 98.1% identity, 98.2% identity, 98.3% identity, 98.4% identity, 98.5% identity, 98.6% identity, 98.7% identity, 98.8% identity, 98.9% identity, 99% identity, 99.1% identity, 99.2% identity, 99.3% identity, 99.4% identity, 99.5% identity, 99.6% identity, 99.7% identity, 99.8% identity, 99.9 or 99.9%.

In one embodiment the genotype 3a is of the strain S52.

In another embodiment the genotype 4a is of the strain ED43.

In one embodiment, the nucleic acid sequences of the invention can be inserted into an expression vector that functions in eukaryotic cells. Eukaryotic expression vectors suitable for producing high efficiency gene transfer in vivo are well known to those of ordinary skill in the art and include, but are not limited to, plasmids, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses.

In another embodiment, the sequences contained in the recombinant expression vector can be transcribed in vitro by methods known to those of ordinary skill in the art in order to produce RNA transcripts, which encode the hepatitis C viruses of the invention. The hepatitis C viruses of the invention may then be produced by transfecting cells by methods known to those of ordinary skill in the art with either the in vitro transcription mixture containing the RNA transcripts or with the recombinant expression vectors containing the nucleic acid sequences described herein.

An embodiment of the present invention relates to a DNA construct comprising a nucleic acid molecule of the present invention.

Another embodiment of the present invention relates to an RNA transcript of the DNA construct comprising a nucleic acid molecule of the present invention.

Infectious Nucleic Acid Sequences and Viruses

The invention further relates to mutations of the infectious nucleic acid sequences of the invention where mutation includes, but is not limited to, point mutations, deletions and insertions.

In one embodiment, a gene or fragment thereof can be deleted to determine the effect of the deleted gene or genes on the properties of the encoded virus such as its virulence and its ability to replicate.

In one embodiment, a gene or fragment can be inserted to determine the effect of the insertion. This insertion could be an HCV genome fragment, but also a heterologous sequence, such as a reporter gene.

In an alternative embodiment, a mutation may be introduced into the infectious nucleic acid sequences to examine the effect of the mutation on the properties of the virus.

The invention also relates to the introduction of mutations or deletions into the infectious nucleic acid sequence in order to produce an attenuated hepatitis C virus suitable for vaccine development.

The invention further relates to the use of the infectious nucleic acid sequences to produce attenuated viruses via passage in vitro or in vivo of the viruses produced by transfection of a host cell with the infectious nucleic acid sequences.

The present invention also relates to the use of the nucleic acid sequences of the invention or fragments thereof in the production of polypeptides where “nucleic acid sequences of the invention” refers to infectious nucleic acid sequences, mutations of infectious nucleic acid sequence, chimeric nucleic acid sequence and sequences which comprise the genome of attenuated viruses produced from the infectious nucleic acid sequence of the invention.

The invention further relates to mutations of the infectious nucleic acid sequences where “mutations” include, but are not limited to, point mutations, deletions and insertions. Of course, one of ordinary skill in the art would recognize that the size of the insertions would be limited by the ability of the resultant nucleic acid sequence to be properly packaged within the virion. Such mutations could be produced by techniques known to those of skill in the art such as site-directed mutagenesis, fusion PCR, and restriction digestion followed by religation.

In one embodiment, mutagenesis might be undertaken to determine sequences that are important for viral properties such as replication or virulence. For example, one may introduce a mutation into the infectious nucleic acid sequence, which eliminates the cleavage site between the NS4A and NS4B polypeptides to examine the effects on viral replication and processing of the polypeptide.

Alternatively, one may delete all or part of a gene or of the 5′ or 3′ untranslated region contained in an infectious nucleic acid sequence and then transfect a host cell (animal or cell culture) with the mutated sequence and measure viral replication in the host by methods known in the art such as RT-PCR. Genes include, but are not limited to, Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B genes but also the untranslated regions. Of course, those of ordinary skill in the art will understand that deletion of part of a gene, preferably the central portion of the gene, may be preferable to deletion of the entire gene in order to conserve the cleavage site boundaries which exist between proteins in the HCV polyprotein and which are necessary for proper processing of the polyprotein.

In the alternative, if the transfection is into a host animal such as a chimpanzee, one can monitor the virulence phenotype of the virus produced by transfection of the mutated infectious nucleic acid sequence by methods known in the art such as measurement of liver enzyme levels (alanine aminotransferase (ALT) or isocitrate dehydrogenase (ICD)) or by histopathology of liver biopsies. Thus, mutations of the infectious nucleic acid sequences may be useful in the production of attenuated HCV strains suitable for vaccine use.

The invention also relates to the use of the infectious nucleic acid sequence of the present invention to produce attenuated viral strains via passage in vitro or in vivo of the virus produced by transfection with the infectious nucleic acid sequence.

In an embodiment of the present invention the molecule of the present invention is capable of expressing HCV when transfected into cells.

In another embodiment of the present invention the molecule of the present invention is capable of infectivity in vivo.

An embodiment of the present invention relates to an in vivo or an in vitro cell transfected with the DNA comprising a nucleic acid molecule of the present invention.

In an embodiment of the present invention these cells are mammalian cells such as human cells.

In an embodiment of the present invention these cells are mammalian cells such as chimpanzee cells.

Another embodiment of the present invention relates to a cell transfected with an RNA transcript of the DNA comprising a nucleic acid molecule of the present invention or an RNA transcript of the nucleic acid molecule of the present invention.

The present invention therefore relates to the use of the nucleic acid sequence of the invention to identify cell lines capable of supporting the replication of HCV.

In particular, it is contemplated that the mutations of the infectious nucleic acid sequence of the invention and the production of chimeric sequences as discussed above may be useful in identifying sequences critical for cell culture adaptation of HCV and hence, may be useful in identifying cell lines capable of supporting HCV replication.

Transfection of tissue culture cells with the nucleic acid sequences of the invention may be done by methods of transfection known in the art such as electroporation, precipitation with DEAE-Dextran or calcium phosphate or liposomes.

In one such embodiment, the method comprises the growing of animal cells, especially human cells, in vitro and transfecting the cells with the nucleic acid of the invention, then determining if the cells show indicia of HCV infection. Such indicia include the detection of viral antigens in the cell, for example, by immunofluorescence procedures well known in the art; the detection of viral polypeptides by Western blotting using antibodies specific therefore; and the detection of newly transcribed viral RNA within the cells via methods such as RT-PCR. The presence of live, infectious virus particles following such tests may also be shown by injection of cell culture medium or cell lysates into healthy, susceptible animals, with subsequent exhibition of the signs and symptoms of HCV infection.

An embodiment of the present invention relates to a method for determining the susceptibility of cells in vitro to support HCV infection, comprising the steps of: growing cells in vitro, transfecting into said cells the nucleic acid of the present invention, and determining if said cells show indicia of HCV replication.

Suitable cells or cell lines for culturing HCV include, but are not limited to, lymphocyte and hepatocyte cell lines known in the art.

In an embodiment of the present invention pertains to cells for culturing HCV—said cells may be used in a method for determining the susceptibility of cells in vitro to support HCV infection are human cells comprising the steps of: a) growing animal cells in vitro; b) transfecting into said cells the nucleic acid according to the present invention and c) determining if said cells show indicia of HCV replication.

Alternatively, primary hepatocytes can be cultured, and then infected with HCV; or, the hepatocyte cultures could be derived from the livers of infected chimpanzees. In addition, various immortalization methods known to those of ordinary skill in the art can be used to obtain cell lines derived from hepatocyte cultures. For example, primary hepatocyte cultures may be fused to a variety of cells to maintain stability.

The present invention further relates to the in vitro and in vivo production of hepatitis C viruses from the nucleic acid sequences of the invention.

An embodiment of the present invention relates to a hepatitis C virus polypeptide produced by a cell transfected with DNA comprising a nucleic acid molecule of the present invention.

Another embodiment of the present invention relates to a hepatitis C virus polypeptide produced by a cell transfected with the RNA transcript of the DNA comprising a nucleic acid molecule of the present invention or the nucleic acid molecule of the present invention.

Yet another embodiment of the present invention relates to a hepatitis C virus produced by a cell transfected with DNA comprising a nucleic acid molecule of the present invention.

An embodiment of the present invention relates to a hepatitis C virus produced by a cell transfected with the RNA transcript of DNA comprising a nucleic acid molecule of the present invention or the nucleic acid molecule of the present invention.

Another embodiment of the present invention relates to a hepatitis C virus whose genome comprises the nucleic acid molecule of the present invention.

An embodiment of the present invention relates to a method for producing a hepatitis C virus comprising transfecting a host cell with the RNA transcript of DNA comprising a nucleic acid molecule of the present invention or the nucleic acid molecule of the present invention, or an RNA transcript of the nucleic acid molecules of the invention.

A further embodiment of the present invention relates to a polypeptide encoded by a nucleic acid sequence of the present invention.

Another embodiment of the present invention relates to a polypeptide encoded by a nucleic acid sequence of the present invention, wherein said polypeptide is selected from the group consisting of Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B.

Chimeric Nucleic Acid Sequences

Nucleic acid sequences, which comprise sequences from two or more HCV genotypes or subtypes are designated “chimeric nucleic acid sequences”. Alternatively, they are designated “intergenotypic recombinants”, if the sequences stem from different HCV genotypes or subtypes; and they are designated “intragenotypic recombinants” if the sequences stem from different isolates/strains of the same genotype subtype.

The invention also relates to “chimeric nucleic acid sequences” or “intra- and intergenotypic recombinant nucleic acid sequences”, where the chimeric nucleic acid sequences consist of open-reading frame sequences and/or 5′ and/or 3′ untranslated sequences taken from nucleic acid sequences of hepatitis C viruses of different genotypes or subtypes or isolates

In one embodiment, the chimeric nucleic acid sequence consists or is comprised of sequences from the genome of infectious HCV of genotype 3a or 4a which encodes structural polypeptides and sequence from the genome of a HCV of a different genotype or subtype which encodes nonstructural polypeptides.

Alternatively, the nonstructural region of infectious HCV of genotypes 3a and 4a and structural region of a HCV of a different genotype or subtype may be combined. This will result in a chimeric nucleic acid sequence consisting of sequence from the genome of infectious HCV of genotype 3a or 4a, which encodes nonstructural polypeptides and sequence from the genome of a HCV of a another genotype or subtype which encodes structural polypeptides.

Alternatively, only one or several structural or non-structural gene from infectious HCV of genotypes 3a and 4a might be inserted into a genome of another HCV isolate. Also, only one or several structural or non-structural gene from another HCV isolate might be inserted into infectious HCV of genotypes 3a and 4a.

Further, only a certain genomic region, not comprising an entire gene of infectious HCV of genotypes 3a and 4a might be inserted into a genome of another HCV isolate. Also only a certain genomic region, not comprising an entire gene from another HCV isolate might be inserted into infectious HCV of genotypes 3a and 4a.

It is believed that the construction of such chimeric nucleic acid sequences will be of importance in studying the growth and virulence properties of hepatitis C virus and in the production of candidate hepatitis C virus vaccines suitable to confer protection against multiple genotypes of HCV. For example, one might produce a “multivalent” vaccine by putting epitopes from several genotypes or subtypes into one clone. Alternatively one might replace just a single gene from an infectious sequence with the corresponding gene from the genomic sequence of a strain from another genotype or subtype or create a chimeric gene, which contains portions of a gene from two genotypes or subtypes. Examples of genes which could be replaced or which could be made chimeric, include, but are not limited to, the E1, E2 and NS4 genes.

Uses of the Nucleic Acid Sequences, Viruses and Polypeptides of the Invention

The hepatitis C viruses produced from the sequences of the invention may be purified or partially purified from the transfected cells by methods known to those of ordinary skill in the art. In a preferred embodiment, the viruses are partially purified prior to their use as immunogens in the pharmaceutical compositions and vaccines of the present invention.

The present invention therefore relates to the use of the hepatitis C viruses produced from the nucleic acid sequences of the invention as immunogens in killed (e.g., formalin inactivated) vaccines to prevent hepatitis C in a mammal.

In an alternative embodiment, the immunogen of the present invention may be an infectious nucleic acid sequence, a chimeric nucleic acid sequence, or a mutated infectious nucleic acid sequence, which encodes a hepatitis C virus. Where the sequence is a cDNA sequence, the cDNAs and their RNA transcripts may be used to transfect a mammal by direct injection into the liver tissue of the mammal as described in the Examples.

Alternatively, direct gene transfer may be accomplished via administration of a eukaryotic expression vector containing a nucleic acid sequence of the invention.

In yet another embodiment, the immunogen may be a polypeptide encoded by the nucleic acid sequences of the invention. The present invention therefore also relates to polypeptides produced from the nucleic acid sequences of the invention or fragments thereof. In one embodiment, polypeptides of the present invention can be recombinantly produced by synthesis from the nucleic acid sequences of the invention or isolated fragments thereof, and purified, or partially purified, from transfected cells using methods already known in the art. In an alternative embodiment, the polypeptides may be purified or partially purified from viral particles produced via transfection of a host cell with the nucleic acid sequences of the invention. Such polypeptides might, for example, include either capsid or envelope polypeptides prepared from the sequences of the present invention.

When used as immunogens, the nucleic acid sequences of the invention, or the polypeptides or viruses produced therefrom, are preferably partially purified prior to use as immunogens in pharmaceutical compositions and vaccines of the present invention. When used as a vaccine, the sequences and the polypeptide and virus products thereof, can be administered alone or in a suitable diluent, including, but not limited to, water, saline, or some type of buffered medium. The vaccine according to the present invention may be administered to an animal, especially a mammal, and most especially a human, by a variety of routes, including, but not limited to, intradermally, intramuscularly, subcutaneously, or in any combination thereof.

Suitable amounts of material to administer for prophylactic and therapeutic purposes will vary depending on the route selected and the immunogen (nucleic acid, virus, polypeptide) administered. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. The vaccines of the present invention may be administered once or periodically until a suitable titer of anti-HCV antibodies appear in the blood. For an immunogen consisting of a nucleic acid sequence, a suitable amount of nucleic acid sequence to be used for prophylactic purposes might be expected to fall in the range of from about 100 μg to about 5 mg and most preferably in the range of from about 500 μg to about 2 mg. For a polypeptide, a suitable amount to use for prophylactic purposes is preferably 100 ng to 100 μg, and for a virus 10² to 10⁶ infectious doses. Such administration will, of course, occur prior to any sign of HCV infection.

A vaccine of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. An inert carrier is preferably used, such as saline or phosphate-buffered saline, or any such carrier in which the HCV of the present invention can be suitably suspended. The vaccines may be in the form of single dose preparations or in multi-dose flasks, which can be utilized for mass-vaccination programs of both animals and humans. For purposes of using the vaccines of the present invention reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (Ed.) (1980); and New Trends and Developments in Vaccines, Voller et al. (Eds.), University Park Press, Baltimore, Md. (1978), both of which provide much useful information for preparing and using vaccines. Of course, the polypeptides of the present invention, when used as vaccines, can include, as part of the composition or emulsion, a suitable adjuvant, such as alum (or aluminum hydroxide) when humans are to be vaccinated, to further stimulate production of antibodies by immune cells. When nucleic acids, viruses or polypeptides are used for vaccination purposes, other specific adjuvants such as CpG motifs (Krieg, A. K. et al. (1995) and (1996)), may prove useful.

When the nucleic acids, viruses and polypeptides of the present invention are used as vaccines or inocula, they will normally exist as physically discrete units suitable as a unitary dosage for animals, especially mammals, and most especially humans, wherein each unit will contain a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent. The dose of said vaccine or inoculum according to the present invention is administered at least once. In order to increase the antibody level, a second or booster dose may be administered at some time after the initial dose. The need for, and timing of, such booster dose will, of course, be determined within the sound judgment of the administrator of such vaccine or inoculum and according to sound principles well known in the art. For example, such booster dose could reasonably be expected to be advantageous at some time between about 2 weeks to about 6 months following the initial vaccination. Subsequent doses may be administered as indicated.

The nucleic acid sequences, viruses and polypeptides of the present invention can also be administered for purposes of therapy, where a mammal, especially a primate, and most especially a human, is already infected, as shown by well-known diagnostic measures. When the nucleic acid sequences, viruses or polypeptides of the present invention are used for such therapeutic purposes, much of the same criteria will apply as when it is used as a vaccine, except that inoculation will occur post-infection. Thus, when the nucleic acid sequences, viruses or polypeptides of the present invention are used as therapeutic agents in the treatment of infection, the therapeutic agent comprises a pharmaceutical composition containing a sufficient amount of said nucleic acid sequences, viruses or polypeptides so as to elicit a therapeutically effective response in the organism to be treated. Of course, the amount of pharmaceutical composition to be administered will, as for vaccines, vary depending on the immunogen contained therein (nucleic acid, polypeptide, virus) and on the route of administration.

The therapeutic agent according to the present invention can thus be administered by subcutaneous, intramuscular or intradermal routes. One skilled in the art will certainly appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Of course, the actual amounts will vary depending on the route of administration as well as the sex, age, and clinical status of the subject which, in the case of human patients, is to be determined with the sound judgment of the clinician.

The therapeutic agent of the present invention can be employed in such forms as capsules, liquid solutions, suspensions or elixirs, or sterile liquid forms such as solutions or suspensions. An inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the HCV of the present invention can be suitably suspended. The therapeutic agents may be in the form of single dose preparations or in the multi-dose flasks, which can be utilized for mass-treatment programs of both animals and humans. Of course, when the nucleic acid sequences, viruses or polypeptides of the present invention are used as therapeutic agents they may be administered as a single dose or as a series of doses, depending on the situation as determined by the person conducting the treatment.

The nucleic acids, polypeptides and viruses of the present invention can also be utilized in the production of antibodies against HCV. The term “antibody” is herein used to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Examples of antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, F(ab′)₂ and F(v) as well as chimeric antibody molecules.

Thus, the polypeptides, viruses and nucleic acid sequences of the present invention can be used in the generation of antibodies that immunoreact (i.e., specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or an active portion thereof) with antigenic determinants on the surface of hepatitis C virus particles.

The present invention therefore also relates to antibodies produced following immunization with the nucleic acid sequences, viruses or polypeptides of the present invention. These antibodies are typically produced by immunizing a mammal with an immunogen or vaccine to induce antibody molecules having immunospecificity for polypeptides or viruses produced in response to infection with the nucleic acid sequences of the present invention. When used in generating such antibodies, the nucleic acid sequences, viruses, or polypeptides of the present invention may be linked to some type of carrier molecule. The resulting antibody molecules are then collected from said mammal. Antibodies produced according to the present invention have the unique advantage of being generated in response to authentic, functional polypeptides produced according to the actual cloned HCV genome.

The antibody molecules of the present invention may be polyclonal or monoclonal. Monoclonal antibodies are readily produced by methods well known in the art. Portions of immunoglobin molecules, such as Fabs, as well as chimeric antibodies, may also be produced by methods well known to those of ordinary skill in the art of generating such antibodies.

The antibodies according to the present invention may also be contained in blood, plasma, serum, hybridoma supernatants, and the like. Alternatively, the antibody of the present invention is isolated to the extent desired by well-known techniques such as, for example, using DEAE Sephadex. The antibodies produced according to the present invention may be further purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, and the like. Antibodies of the IgG class are preferred for purposes of passive protection.

The antibodies of the present invention are useful in the prevention and treatment of diseases caused by hepatitis C virus in animals, especially mammals, and most especially humans. Examples for indications of antibody treatment are post-exposure prophylaxis after needle-stick injuries or re-infection prophylaxis after liver transplantation.

An embodiment of the present invention relates to an antibody to the polypeptide encoded by the nucleic acid sequences of the present invention.

An embodiment of the present invention relates to an antibody to the hepatitis C virus produced from the nucleic acid sequences of the present invention.

In providing the antibodies of the present invention to a recipient mammal, preferably a human, the dosage of administered antibodies will vary depending on such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, and the like.

In general, it will be advantageous to provide the recipient mammal with a dosage of antibodies in the range of from about 1 mg/kg body weight to about 10 mg/kg body weight of the mammal, although a lower or higher dose may be administered if found desirable. Such antibodies will normally be administered by intravenous or intramuscular route as an inoculum. The antibodies of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, lessen or attenuate the severity, extent or duration of any existing infection.

The antibodies prepared by use of the nucleic acid sequences, viruses or polypeptides of the present invention are also highly useful for diagnostic purposes. For example, the antibodies can be used as in vitro diagnostic agents to test for the presence of HCV in biological samples taken from animals, especially humans. Such assays include, but are not limited to, radioimmunoassays, EIA, fluorescence, Western blot analysis and ELISAs. In one such embodiment, the biological sample is contacted with antibodies of the present invention and a labeled second antibody is used to detect the presence of HCV to which the antibodies are bound.

Such assays may be, for example, direct where the labeled first antibody is immunoreactive with the antigen, such as, for example, a polypeptide on the surface of the virus; indirect where a labeled second antibody is reactive with the first antibody; a competitive protocol such as would involve the addition of a labeled antigen; or sandwich where both labeled and unlabeled antibody are used, as well as other protocols well known and described in the art.

In one embodiment, an immunoassay method would utilize an antibody specific for HCV envelope determinants and would further comprise the steps of contacting a biological sample with the HCV-specific antibody and then detecting the presence of HCV material in the test sample using one of the types of assay protocols as described above. Polypeptides and antibodies produced according to the present invention may also be supplied in the form of a kit, either present in vials as purified material, or present in compositions and suspended in suitable diluents as previously described.

In a preferred embodiment, such a diagnostic test kit for detection of HCV antigens in a test sample comprises in combination a series of containers, each container a reagent needed for such assay. Thus, one such container would contain a specific amount of HCV-specific antibody as already described, a second container would contain a diluent for suspension of the sample to be tested, a third container would contain a positive control and an additional container would contain a negative control. An additional container could contain a blank.

For all prophylactic, therapeutic and diagnostic uses, the antibodies of the invention and other reagents, plus appropriate devices and accessories, may be provided in the form of a kit so as to facilitate ready availability and ease of use.

The present invention also relates to the use of nucleic acid sequences and polypeptides of the present invention to screen potential antiviral agents for antiviral activity against HCV. Such screening methods are known by those of skill in the art. Generally, the antiviral agents are tested at a variety of concentrations, for their effect on preventing viral replication in cell culture systems, which support viral replication, and then for an inhibition of infectivity or of viral pathogenicity (and a low level of toxicity) in an animal model system.

In one embodiment, animal cells (especially human cells) transfected with the nucleic acid sequences of the invention are cultured in vitro and the cells are treated with a candidate antiviral agent (a chemical, peptide etc.) by adding the candidate agent to the medium. The treated cells are then exposed, possibly under transfecting or fusing conditions known in the art, to the nucleic acid sequences of the present invention. A sufficient period of time would then be allowed to pass for infection to occur, following which the presence or absence of viral replication would be determined versus untreated control cells by methods known to those of ordinary skill in the art. Such methods include, but are not limited to, the detection of viral antigens in the cell, for example, by immunofluorescence procedures well known in the art; the detection of viral polypeptides by Western blotting using antibodies specific therefore; the detection of newly transcribed viral RNA within the cells by PT-PCR; and the detection of the presence of live, infectious virus particles by injection of cell culture medium or cell lysates into healthy, susceptible animals, with subsequent exhibition of the signs and symptoms of HCV infection. A comparison of results obtained for control cells (treated only with nucleic acid sequence) with those obtained for treated cells (nucleic acid sequence and antiviral agent) would indicate, the degree, if any, of antiviral activity of the candidate antiviral agent. Of course, one of ordinary skill in the art would readily understand that such cells can be treated with the candidate antiviral agent either before or after exposure to the nucleic acid sequence of the present invention so as to determine what stage, or stages, of viral infection and replication said agent is effective against.

In an alternative embodiment, viral enzyme such as but not exclusively the NS3 protease, NS2-NS3 autoprotease, NS3 helicase, NS4A (NS3 protease co-factor), NS5A or NS5B RNA polymerase may be produced from a nucleic acid sequence of the invention and used to screen for inhibitors, which may act as antiviral agents. The E1/E2 envelope proteins may be produced to evaluate the function of entry inhibitors in certain laboratory assays. The structural and nonstructural regions of the HCV genome, including nucleotide and amino acid locations, have been determined.

Such above-mentioned protease inhibitors may take the form of chemical compounds or peptides, which mimic the known cleavage sites of the protease and may be screened using methods known to those of skill in the art. For example, a substrate may be employed which mimics the protease's natural substrate, but which provides a detectable signal (e.g. by fluorimetric or colorimetric methods) when cleaved. This substrate is then incubated with the protease and the candidate protease inhibitor under conditions of suitable pH, temperature etc. to detect protease activity. The proteolytic activities of the protease in the presence or absence of the candidate inhibitor are then determined.

In yet another embodiment, a candidate antiviral agent (such as a protease inhibitor) may be directly assayed in vivo for antiviral activity by administering the candidate antiviral agent to a chimpanzee transfected with a nucleic acid sequence of the invention or infected with a virus of the invention and then measuring viral replication in vivo via methods such as RT-PCR. Of course, the chimpanzee may be treated with the candidate agent either before or after transfection with the infectious nucleic acid sequence or infected with a virus of the invention so as to determine what stage, or stages, of viral infection and replication the agent is effective against.

An embodiment of the present invention relates to a method for assaying candidate antiviral agents for activity against HCV, comprising: exposing a cell containing the hepatitis C virus produced by the nucleic acid sequences of the present invention to the candidate antiviral agent; and measuring the presence or absence of hepatitis C virus replication or correlates thereof in said cell.

An embodiment of the present invention relates to a method for assaying candidate antiviral agents for activity against HCV, comprising: exposing a cell containing the hepatitis C virus produced by the nucleic acid sequences of the present invention to the candidate antiviral agent; and measuring the presence or absence of hepatitis C virus replication or correlates thereof in said cell by at least one of the following: negative strand RT-PCR, quantitative RT-PCR, Western blot, immunofluorescence, non-fluorescent immuno-staining, or infectivity in a susceptible animal.

An embodiment of the present invention relates to an antiviral agent identified as having antiviral activity for HCV by the methods for assaying candidate antiviral agents for activity against HCV.

The invention also provides that the nucleic acid sequences, viruses and polypeptides of the invention may be supplied in the form of a kit, alone or in the form of a pharmaceutical composition.

In one embodiment, said polypeptide or polypeptides are fully or partially purified from hepatitis C virus produced by cells transfected with nucleic acid sequence of the invention.

In another embodiment, the polypeptide or polypeptides are produced recombinantly from a fragment of the nucleic acid sequences of the invention.

In yet another embodiment, the polypeptides are chemically synthesized.

The polypeptides of the invention, especially structural polypeptides, can serve as immunogens in the development of vaccines or as antigens in the development of diagnostic assays for detecting the presence of HCV in biological samples.

The invention therefore also relates to vaccines for use in immunizing mammals especially humans against hepatitis C. In one embodiment, the vaccine comprises one or more polypeptides made from the nucleic acid sequence of the invention or fragment thereof. In a second embodiment, the vaccine comprises a hepatitis C virus produced by transfection of host cells with the nucleic acid sequences of the invention.

The present invention therefore relates to methods for preventing hepatitis C in a mammal.

In one embodiment the method comprises administering to a mammal a polypeptide or polypeptides encoded by the nucleic acid sequence of the invention in an amount effective to induce protective immunity to hepatitis C.

In another embodiment, the method of prevention comprises administering to a mammal a hepatitis C virus of the invention in an amount effective to induce protective immunity against hepatitis C.

In yet another embodiment, the method of protection comprises administering to a mammal the nucleic acid sequence of the invention or a fragment thereof in an amount effective to induce protective immunity against hepatitis C.

The invention also relates to hepatitis C viruses produced by host cells transfected with the nucleic acid sequence of the present invention.

The invention therefore also provides pharmaceutical compositions comprising the nucleic acid sequence of the invention and/or the encoded hepatitis C viruses. The invention further provides pharmaceutical compositions comprising polypeptides encoded by the nucleic acid sequence of the invention or fragments thereof. The pharmaceutical compositions of the invention may be used prophylactic or therapeutically.

An embodiment of the present invention relates to a composition comprising a polypeptide encoded by the nucleic acid sequences of the present invention suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.

An embodiment of the present invention relates to a composition comprising a nucleic acid molecule of the present invention suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.

The invention also relates to antibodies to the hepatitis C virus of the invention or their encoded polypeptides and to pharmaceutical compositions comprising these antibodies.

The invention also relates to the use of the nucleic acid sequences of the invention to identify cell lines capable of supporting the replication of HCV in vitro.

The invention further relates to the use of the nucleic acid sequences of the invention or their encoded viral enzymes (e.g. NS3 serine protease, NS3 helicase, NS4A, NS5A, NS5B RNA polymerase) to develop screening assays to identify antiviral agents for HCV.

An embodiment of the present invention relates to a method for assaying candidate antiviral agents for activity against HCV, comprising: exposing an HCV protease encoded by a nucleic acid sequence of the present invention or a fragment thereof to the candidate antiviral agent in the presence of a protease substrate; and measuring the protease activity of said protease.

An embodiment of the present invention relates to a method for assaying candidate antiviral agents for activity against HCV, comprising: exposing an HCV protease encoded by a nucleic acid sequence of the present invention or a fragment thereof to the candidate antiviral agent in the presence of a protease substrate; and measuring the protease activity of said protease, wherein said HCV protease is selected from the group consisting of an NS3 domain protease, an NS3-NS4A fusion polypeptide, or an NS2-NS3 protease.

An embodiment of the present invention relates to an antiviral agent identified as having antiviral activity for HCV by the method assaying candidate antiviral agents for activity against HCV.

All scientific publication and/or patents cited herein are specifically incorporated by reference. The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

FIGURES

FIG. 1

Phylogenetic Tree of pS52, pED43 and Representative HCV cDNA Clones and Isolates of HCV Genotypes 1-4

Genbank accession numbers and names of isolates/clones (in parenthesis) are given. Multiple polyprotein sequence alignment and neighbor-joining tree analysis using amino acid p-distance model were done with Mega4.1. (*) Infectious cDNA clones.

FIG. 2

Course of Infection with HCV Following Intrahepatic Transfection of Chimpanzees 5276 (FIG. 2A) and 5300 (FIG. 28) with RNA Transcripts of pS52 (genotype 3a) and pED43 (Genotype 4a), Respectively.

Serum samples collected weekly were tested for HCV-RNA by in-house Taqman assay (detection limit of 10 IU/ml) and/or by the Roche Monitor Test 2.0 (detection limit of 600 IU/ml): filled rectangle, positive by Taqman and/or by Monitor; empty rectangle, negative by Taqman. Black dots, HCV Monitor titers—samples below the detection limit are shown as not detected (ND). Anti-HCV antibodies were detected in the 2nd generation ELISA: +, positive; − negative. Shaded area: serum ALT (U/L). Weekly liver biopsies were collected and examined for necro-inflammatory changes: 0, normal; 1, mild; 2, mild-moderate; 3, moderate-severe or 4, severe. The nucleotide sequence of the entire open reading frame of recovered virus genomes in CH5276 (at weeks 7 and 10; open arrows) and CH5300 (at weeks 1 and 6; open arrows) were identical with the sequence of pS52 and pED43, respectively.

Serum Neutralizing antibodies: Percent neutralization of JFH1-based intergenotypic recombinants expressing the S52 (A) or ED43 (B) envelope proteins (>50% considered significant). Percent neutralization of 1:20 serum dilutions was determined by comparison with replicates of weeks −1 and 0, as described in Materials and Methods below. Values represent the mean of three neutralizations; SEM ranged from 3-13% and 1-16% for the S52 (A) and ED43 (B), respectively. Negative values are shown as 0%. We also tested 1:80 serum dilutions; in all cases percent neutralization was <20%.

Peripheral and intrahepatic CD4+/CD8+ T-cell responses: Amount of IFN-γ secreting cells after stimulation with a panel of overlapping peptides, spanning the entire HCV polyprotein, in ELISpot assays. Genotype 3a (strain K3a/650) specific peptides were used for testing of T cells derived from CH5276 (A) and genotype 4a (strain ED43) specific peptides were used for testing of T cells derived from CH5300 (B). PBMC were used directly. Intrahepatic CD4+ and CD8+ T cells were expanded from liver biopsies as described in Materials and Methods. Heights of bars represent the total number of IFN-γ secreting CD4+ and CD8+ T cells following stimulation with the different pools, after background subtraction. The number of IFN-γ secreting cells seen after stimulation with individual peptide pools is colour coded. Cut-off points were determined for individual experiments as described in Materials and Methods. ND, not determinable, visualized by dotted line; results below cut-off are indicated by black bars up to the dotted line.

FIG. 3

Genetic Heterogeneity of S52 Virus Population from Chimpanzee Acute Phase Plasma Pool

(1) Nt and aa positions refer to pS52. Number (#) and percentage (%, related to respective genomic region) of positions with genetic heterogeneity (GH) in the analysed clones; clonal analysis was done as described in Materials and Methods. GH≧1 clone and GH≧2 clones; GH at the respective position found in at least 1 or in at least 2 of the analysed clones. Defective genomes were not considered to reflect naturally occurring genetic heterogeneity and are therefore not included; in one of the clones analysed at the respective position nt change G5618A introduced a stop codon and nt insertions at position 6761.1 and 9118.1 caused frame shifts.

FIG. 4

Comparison of ORF Sequence of S52 with that of Other Genotype 3a Isolates

(1) Nt and aa positions refer to pS52. Number (#) and percentage (%, related to respective genomic region) of positions with sequence variation between S52 consensus sequence and published sequences with complete ORF of other genotype 3a isolates. At nt 5358, 3/6 S52 clones had T, the other 3 had C, while all other genotype 3a isolates had either T or C. Thus, this position was not considered to be different.

FIG. 5

Genetic Heterogeneity of ED43 Virus Population from Chimpanzee Acute Phase Plasma Pool

(1) Nt and aa positions refer to pED43. Number (#) and percentage (%, related to respective genomic region) of positions with genetic heterogeneity (GH) in the analysed clones. GH≧1 clone and GH≧2 clones; GH at the respective position found in at least 1 or in at least 2 of the analysed clones. A stop codon (resulting from nt change at position 1930 in one clone) was not considered to reflect naturally occurring genetic heterogeneity and is not included in this analysis.

FIG. 6

Comparison of ORF Sequence of ED43 Derived from Chimpanzee Plasma Pool with ORF of Other Genotype 4a Isolates

(1) Nt and aa positions refer to pED43. Number (#) and percentage (%, related to respective genomic region) of positions with sequence variation between ED43 consensus sequence and published sequences of complete ORF of other genotype 4a isolates. At all positions, at which no distinct nt and/or aa consensus was provided, genetic variation was only assumed, if the provided information clearly showed a difference (e.g. at nt 1966 G and A were found for ED43; thus genetic variation was assumed for another isolate, if T or C was found at the respective position).

FIG. 7

3′ UTR Variable Region of pS52 and Other Genotype 3a Isolates

Variable 3′ UTR in different pS52 and other genotype isolates; n.a., no isolate name assigned.

FIG. 8

3′ UTR Variable Region of pED43 and Other Genotype 4a Isolates

Variable 3′ UTR in different pED43 and other genotype isolates; n.a., no isolate name assigned.

EXAMPLES

Materials and Methods

Source of HCV strains S52 and ED43. Genotype 3a strain S52 and genotype 4a strain ED43 were derived from challenge plasma pools from chimpanzees, experimentally infected with serum from chronically infected patients.

Amplification, Cloning and Sequence Analysis

RNA was extracted from 200 ul of the S52 or ED43 plasma pool, respectively, with High Pure Viral Nucleic Acid Kit (Roche) or TRIzol (Invitrogen). cDNA was synthesized with SuperScript II or III (Invitrogen) and random hexamers or specific reverse primers (TAG Copenhagen). After treatment of cDNA with RNase H (Invitrogen) and RNase T1 (Ambion), PCR was carried out with BD Advantage 2 Polymerase Mix (Clontech); PCR of 3′ UTR fragments was carried out with Amplitaq Gold DNA polymerase (Applied Biosystems). Gel purified amplicons were A-tailed with Taq DNA polymerase (Invitrogen), cloned in pCR2.1-Topo or pCR-XL-TOPO (Invitrogen) and transformed in Top10 chemically competent bacteria (Invitrogen). In addition, S52 and ED43 3′ UTR amplicons were subcloned after restriction digest. Sequence analysis and determination of consensus sequence was done using Sequencher, Gene Codes Corporation and freeware BioEdit.

Polyprotein alignments and phylogenetic analysis was done using MEGA4.1 freeware. HCV sequences used for alignments were from the European HCV database website (euHCVdb and the American HCV database website). Standard molecular techniques, such as restriction digest based cloning and fusion PCR, were used for cloning; all fusion PCR were done with Pfu DNA polymerase (Stratagene).

Sequences of strain S52 were obtained by analysis of four amplicons: (i) nt 24 to 3396, (ii) nt 3359 to 5186, (iii) nt 5065 to 7596, and (iv) nt 7530 to 9401. These amplicons covered (i) aa 1-1019, (ii) aa 1008-1715, (iii) aa 1576-2419, and (iii) aa 2398-3020 on the polyprotein (nt and aa numbers refer to positions on pS52 with nt 1 being the 1st nt of the 5′ UTR and aa 1 being the 1st aa of the polyprotein; they do not include primer sequences). Another amplicon (v) contained the C-terminal NS5B sequence (starting from nt 9339) as well as the 3′ UTR variable region, poly-(U/UC) region and the first 16 nt of the conserved X region, and was obtained as previously described; this amplicon covered aa 3001-3021 of the polyprotein sequence. After subcloning, 5 clones of amplicon (i), (ii) and (iv), 6 clones of amplicon (iii), and 15 clones of amplicon (v) were sequenced to determine the consensus sequence. At nt positions 1548 in clone A21 (amplicon i) and 5784 in clone C11 (amplicon iii), the nt was not defined; however, at these positions all other clones analysed had the same nt. pS52 was constructed using clones derived from fragment (i)-(iv), a synthetic 3′ UTR sequence (Genscript) and pGEM-9Zf-MOD. pGEM-9Zf-MOD was generated by replacement of the NotI/EcoRI fragment containing the HCV H77 sequence in pCV-H77C (Yanagi 1997) by a convenient multiple cloning site. In pS52, the NotI site is located immediately upstream of the T7 promoter sequence and the C-terminal XbaI site is located immediately upstream of a AscI site.

For ED43, 5′ UTR and ORF sequences were obtained by two amplicons: (i) nt 28 to 5631, and (ii) nt 5476 to 9376, which covered (i) aa 1-1763 and (ii) aa 1713-3008 (numbers refer to positions on pED43). Another amplicon (iii), spanning the C-terminal NS5B sequence (starting from nt 9301), the 3′ UTR variable region, the poly-(U/UC) region, and the first 16 nt of the conserved X region, was obtained as previously described (Yanagi 1997); this amplicon covered aa 2988-3008. After subcloning, 4 clones of amplicon (i), 5 clones of amplicon (ii), and 10 clones of amplicon (iii) were sequenced to determine the consensus sequence. pED43 was constructed by using clones derived from fragment (i)-(iii) inserted into pCV-H77C (Yanagi 1997) using NotI and NheI sites thereby retaining the 3′ terminal sequence from pCV-H77C (Yanagi 1997). Endotoxin free maxipreps (Quiagen) were prepared and the HCV sequence was confirmed for pS52 and pED43.

Sequencing of Cell Culture Derived HCV

The consensus sequence of the entire ORF of S52 or ED43 genomes recovered from serum of infected chimpanzees was determined by direct sequence analysis of PCR amplicons obtained in a nested RT-PCR procedure.

RNA was extracted from serum using the High Pure Viral Nucleic Acid Kit (Roche) according to manufacturer's protocol. Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using RNA extracted from 100 μL serum. Primers (TAG Copenhagen) were 1.25 μM and dNTPs (Invitrogen) were 0.5 mM in RT reactions. For denaturation, RNA was incubated for 2′ at 65° C. together with primer and dNTPs and placed on ice. cDNA syntheses was done in a 20 μL volume with SuperScriptIII (Invitrogen). The final RT reaction was treated with 1-4 U RNase H (Invitrogen) and 1000 U RNase T1 (Ambion) for 20′ at 37° C. to degrade RNA. 1st round PCR was performed in a 50 μL volume on 2.5 μL of the cDNA reaction using the Advantage 2 PCR Enzyme System (Clontech). Cycle parameters were 5 cycles of 35″ at 99° C., 30″ at 67° C. and 10′ at 68° C., 10 cycles of 35″ at 99° C., 30″ at 67° C. and 11′ at 68° C., 10 cycles of 35″ at 99° C., 30″ at 67° C. and 12′ at 68° C. and 10 cycles of 35″ at 99° C., 30″ at 67° C. and 13′ at 68° C. Several overlapping ˜1 kb products were synthesized in a nested PCR covering the entire ORF. PCR was set up as above using 2.5 μL of the 1st round PCR for each reaction.

Initial denaturation was 35 sec at 99° C. followed by 35 cycles with 35 sec at 99° C., 30 sec at 67° C. and 6 min at 68° C.

Sequencing, Sequence Analysis and Databases

All sequence reactions were carried out at Macrogen Inc., Seoul, South Korea. Sequence analysis was carried out with Sequencher 4.7, Gene Codes Corporation and freeware BioEdit v. 7.0.5. HCV sequences used for alignments were retrieved from The European HCV database (accessible on the internet site euHCVdbhttp at “euhcvdb.ibcp.fr/euHCVdb/”) and the American HCV database (LANL; accessible at the http internet site “hcv.lanl.gov/content/hcv-db/index”).

Generation of RNA Transcripts and Transfections

Plasmid DNA was linearized with XbaI (New England BioLabs) and purified (Wizard SV Gel and PCR Clean-Up System; Promega). 5 mg linearized DAN was in vitro transcribed with T7 RNA Polymerase fro 2 hrs in a final volume of 100 μl, following manufacturer's instructions (Promega). Before generation of RNA transcripts to be used for in vitro transfection, XbaI digested pED43 with and without adaptive mutations was in addition treated with Mung bean nuclease. The amount of RNA transcripts was estimated by standard agarose gel electrophoresis.

For in vitro transfections, Huh7.5 cells were plated at 4×10⁵ per well of a 6-well plate in Dulbecco's modified Eagle medium with 4500 mg/L glucose, GlutaMAX-I, and Pyruvate (Gibco/Invitrogen Corporation) containing 10% heat-inactivated fetal bovine serum (Sigma), penicillin 100 U/mL and streptomycin 100 μg/mL (Gibco/Invitrogen Corporation), at 5% CO2 and 37° C. After 12-24 hrs, cells were incubated with lipofection complexes (RNA transcripts and 5 μL Lipofectamine 2000 [Invitrogen]) in serum-free medium (Opti-MEM; Invitrogen) for approximately 16 hrs.

For in vivo transfections, chimpanzees were housed in compliance with relevant guidelines and requirements, in facilities fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. CH5276 and CH5300 were inoculated intrahepatically by a percutaneous procedure by RNA transcribed as described above from a total of 20 μg XbaI digested and purified pS52 and pED43, respectively.

Monitoring of HCV Infection in Huh7.5 Cells

Huh7.5 cells were immunostained for HCV Core antigen using the primary antibody mouse anti-HCV core protein monoclonal antibody (B2) (Anogen, Yes Biotech Laboratories) at 1:200 in PBS with 5% bovine serum albumin, and the secondary antibody Alexa Fluor 594 goat anti-mouse IgG (H_L) (Invitrogen) at 1:500 in PBS/Tween; cell nuclei were counterstained with Hoechst 33342 (Invitrogen). The presence of HCV-positive cells was evaluated by fluorescence confocal microscopy. Staining was visualized using a Leica TCS confocal microscope. Mouse anti-HCV core protein monoclonal antibody (B2) was shown to readily recognize S52 and ED43 Core proteins.

Monitoring of HCV Infection in Chimpanzees

Pre-infection sera were obtained at weeks 0, −1, −5 and −39 for CH5276 and at weeks 0, −1, −5 and −16 for CH5300; pre-infection liver biopsies were obtained at weeks −1 and −5 for both animals. For CH5276, serum and liver biopsies were collected weekly during weeks 1-32. For CH5300, serum and liver biopsies were taken weekly during weeks 1-18, and every two weeks during weeks 20-32. Thereafter, both animals were followed monthly until week 54 to determine the final outcome of infection. Serum samples were tested for HCV RNA (In House Taqman 14 and Monitor 2.0; Roche Diagnostics), HCV antibodies (ELISA 2.0; Abbott), and alanine aminotransferase (ALT) (Anilytics). Liver biopsy samples were examined for necro-inflammatory changes.

Investigation of Chimpanzee Neutralizing Serum Antibodies

Neutralization assays are known in the art. Briefly, heat-inactivated CH5276 sera were pre-incubated with ˜20 focus forming units (FFU) S52/JFH11793S, K1404Q (Gottwein 2007) and CH5300 sera were pre-incubated with ˜45 FFU ED43/JFH1T827A, T977S (Scheel 2008) for 1 hour at 37° C., followed by 3 hours incubation on 6000 Huh7.5 cells. After 48 hours incubation, cultures were immunostained for HCV NS5A with primary antibody 9E10 (gift from C. Rice), used at 1:1000 in PBS/0.1% tween-20 over night at 4° C. After washing, a 1:300 dilution of 2° Ab HRP-goat anti-mouse IgG (H+L) (Amersham Biosciences) in PBS/0.1% tween-20 was added and incubated for 30′ at room temperature. Staining was developed using DAB substrate kit (DAKO) for 30′ after washing. The number of focus forming units (FFU; cluster of infected cells, separated from adjacent clusters of infected cells by at least 2 uninfected cells) was determined on an ImmunoSpot Series 5 UV Analyzer (CTL Europe GmbH) with customized software kindly provided by Alexey Karulin and Paul Lehmann. From FFU counts in experimental wells, the mean of spot counts of 24 negative control wells was subtracted (˜5 spots for the genotype 3 and 4 neutralization experiments). Count numbers were comparable to manual counting, and in general counts of up to 200 FFU/well were considered reliable, because they were in the linear range of dilution series, carried out in an establishment phase. For CH5276, FFU counts ranged from 26 to 73 FFU/well; for CH5300 counts ranged from 59-146 FFU/well. Percentages of neutralization were obtained by comparison with the mean of FFU counts from all wells, in which the respective virus had been pre-incubated with serum samples from week −1 or from week 0.

Investigation of Chimpanzee Cellular Immune Responses

CD4+/CD8+ T cells were isolated from peripheral blood and from the liver. T cells from liver were expanded in vitro before further analysis. Number of interferon-gamma (IFN-γ) secreting T cells was evaluated in ELISpot (U-Cytech) assays after stimulation with HCV peptides. Synthetic peptides, specific for genotype 3a (strain K3a/650) and 4a (strain ED43), that were approximately 20 aa in length, overlapping by 10 residues, and spanning the entire HCV polyprotein were used. These peptides were assembled in 9 pools and used for stimulation.

Example 1

Genetic Analysis of Genotype 3a Strain S52

The HCV source was from an acute-phase challenge plasma pool from a chimpanzee, experimentally infected with serum from a chronically infected Italian patient. In this pool, the HCV RNA titer was 10^(4.3) IU/ml and the infectious titer was 10³ chimpanzee infectious doses (CID)/ml.

The S52 consensus sequence was determined by clonal sequence analysis of five overlapping RT-PCR amplicons, spanning the complete ORF and partial UTRs as described in Material and Methods. At each nt position, 5-11 clones were analysed. In the 5′ UTR sequence of S52, spanning nt 24-339 (all nt positions refer to final pS52 sequence), genetic heterogeneity among the analysed clones was found at 3 nt positions (with 1 clone being different from the other 4 at each position) (FIG. 3).

The S52 ORF consisted of 9063 nt (nt 340-9402), encoding a 3021 aa polyprotein, followed by a single stop codon (nt 9403-9405). Genetic heterogeneity, with at least one of the analysed clones being different from the S52 consensus sequence, was found at 199 nt positions (2.2%) and 67 aa positions (2.2%) (FIG. 3). At 63 nt (0.7%) and 23 aa (0.8%) positions, at least two clones, covering the respective position, deviated from the S52 consensus sequence.

Compared to the entire polyprotein, a high percentage of aa positions with genetic heterogeneity was found in E1, E2, p7, NS2 and NS5A (FIG. 3). The amino acid sequence of E2 HVR1 was identical between the clones. Amino acid positions with genetic heterogeneity are summarized in Table 1. There was evidence of 2 different S52 quasispecies populations (Table 1). For each sequenced clone, differences to the consensus sequence were found in average at 0.48% of positions at the nucleotide level, and 0.54% of positions at the amino acid level. A defective ORF was found in 3 clones (FIG. 3, Table 1). At nt position 5358, no distinct nt consensus could be determined, since 3 of 6 clones had T, the other 3 had C, with T and C encoding the same aa.

The length of S52 3′ UTR variable region was difficult to define. Even though there was a consensus ACACUCC motif (nt 9418-9424), as described for other isolates 33, a UG dinucleotide, typically preceding the start of the poly(U/UC region) was only found in 1/15 clones analysed. The first 23 nt of the variable region (nt 9403-9425) were identical in the 15 clones. They were followed by a UUC motif (nt 9426-9428), present in 13/15 clones (a comparison of the pS52 3′ UTR variable region to other 3a sequences is shown in FIG. 7). Assuming a variable region of 26 nt (nt 9403-9428), the length of the poly(U/UC) region, which could be determined in 3/15 clones, was 108, 111, and 123 nt, respectively. The first 16 nt of the 3′ UTR X region were identical in all analysed clones.

Compared to 2 other genotype 3a 5′ UTR sequences (genbank accession numbers D17763 and D28917; in the following sections, HCV isolates will be identified by their accession numbers), the obtained S52 consensus sequence showed differences at 1 and 3 nt positions, respectively. In comparison to the partial 5′ UTR and complete Core/E1 sequence (nt 58-1488) obtained from the source patient, the S52 consensus sequence obtained in this study differed at 1 nt/aa position in E1. The S52 consensus ORF differed from 3 published genotype 3a isolates with reported ORF in 4.8-6.5% of positions at the nt level and in 3.6-5.9% of positions at the aa level (FIG. 4). A phylogenetic analysis of the polyprotein of developed HCV cDNA clones and representative HCV isolates showed that pS52 clustered with other genotype 3a isolates (FIG. 1).

Comparing genotype 3a 3′ UTR variable regions, the consensus sequence of the first 23 nt of the S52 3′ UTR (nt 9403-9425) was identical to the equivalent sequence of two other genotype 3a isolates with genebank accession numbers D28917 71 and AF009075, but differed at 1 nt from D17763, and at 3 nt from D85024 and D85025. The consensus UUC (nt 9435-9428), occurring in S52, was also present in D28917; in the other isolates it was replaced by either UUUC or AUUC. The length of the 3′ UTR variable region of other genotype 3a isolates was previously defined to be 28-35 nt, determined by a UG motif not occurring for S52 (FIG. 7). Length of the 3′ UTR poly(U/UC) tract was 110 nt for AF009075 33, and 84 and 86 nt for D85024 and D8502572, respectively. S52 consensus of the first 16 nt of the 3′ UTR X region was identical to genotype 3a isolates AF009075, D85024 and, D85025 and genotype 1a cDNA clone pCV-H77C (AF011751) (Yanagi 1997).

Example 2

Generation of Consensus Clone pS52

The consensus full-length cDNA clone pS52 was constructed in vector pGEM-9Zf as described in Material and Methods. The S52 sequence contained the following structural elements: (I) 5′ UTR of 339 nt, in which nt 24-339 were the S52 consensus sequence, while nt 1-23 were deduced from published genotype 3a 5′ UTR sequences (D28917, D17763). For nt 1, at which G (D28917) and A (D17763) occurred, G was chosen to facilitate in vitro transcription. (II) ORF of 9063 nt (nt 340-9402) with two coding nt changes, G1037A and G1913A, in comparison to the S52 consensus sequence. However, at both positions, A encoded by pS52 was present in 2/5 clones analysed. In addition, in 10 clones of a Core-E2 amplicons generated previously, A was present at position 1037 in 8 clones and at position 1913 in 6 clones. Non-coding nt changes compared to the S52 consensus sequence were A639G, A915T, C1488T, G1575A, C1707T, C2655T, C2805T, C3069T, G3792A, T5187C, T7755A, T8469C, G8745A. Non-coding nt changes A915T and T7755A were inserted to remove consensus XbaI sites, at both positions being present in 5/5 clones analysed. All other non-coding nt changes in pS52 were occurring in at least 1 of the 5 to 6 clones covering the respective position. At position 5358, at which no definite nt consensus was determined, T was introduced in pS52. (III) 3′ UTR of 235 nt (nt 9403-9637) with a variable region of 26 nt (nt 9403-9428), identical to the S52 nt consensus sequence; with a poly(U/UC) region of 111 nt (nt 9429-9539), chosen from one of the 3 clones, in which this region could be entirely sequenced; and with a conserved X region of 98 nt (nt 9540-9637), determined by the pCV-H77C (Yanagi 1997) sequence. The X region from 2 genotype 3a isolates (D85024, D85025) was identical to the pCV-H77C X region, whereas genotype 3a isolate AF009075 differed at nt position 9594 and 9635. An XbaI-site was inserted immediately downstream of the HCV 3′ UTR, for generation of the exact HCV 3′end.

Example 3

Genetic Analysis of Genotype 4a Strain ED43

The HCV source was an acute-phase challenge plasma pool from a chimpanzee, experimentally infected with serum from a chronically infected Egyptian patient. This plasma pool had an HCV RNA titer of 10^(5.5) IU/ml and an infectivity titer of 10⁵ CID/ml. Previously, the complete ORF of the source patient's virus has been sequenced. Furthermore, the complete 3′ UTR of the patient's virus has been sequenced previously.

In the present study, ED43 consensus sequence from the chimpanzee plasma pool was determined by clonal sequence analysis of three overlapping RT-PCR amplicons spanning the complete ORF, and partial UTRs as described in Material and Methods. In ED43 5′ UTR sequences, from nt 28 to nt 340 (nt positions refer to pED43), genetic heterogeneity among 4 clones was found at 6 nt positions (with one clone differing from the other clones at each position) (FIG. 5).

In agreement with the patient's virus sequence 9, ED43 ORF was found to consist of 9024 nt (nt 341-9364), coding for 3008 aa, and terminated by two stop codons (nt 9365-9367 and 9374-9376). Genetic heterogeneity, with at least one of the analysed clones deviating from the ED43 consensus sequence, was found at 144 nt positions (1.6%) and 64 aa positions (2.1%) (FIG. 5). Genetic heterogeneity with at least two clones deviating from the consensus sequence was found at only 3 nt and none of the aa positions.

Compared to the average for the entire polyprotein, on the aa level genetic heterogeneity was relatively high in Core, E1, p7, NS2, NS3, and NS4A. The nt and aa sequence of HVR1 was identical between the clones. AA positions at which individual clones differed from the ED43 consensus sequence are shown in Table 2. For each sequenced clone, quasispecies were found in average at 0.35% at the nt level, and 0.45% at the aa level compared to the consensus sequence. One defective genome was identified (FIG. 5). No distinct consensus could be determined at nt positions 1966 (G/A), 1999 (C/T), 3751 (A/G), and 3871 (C/T) where 2 clones had one nt and 2 clones another nt; these nt changes were all non-coding.

ED43 3′ UTR variable region of 36 nt (nt 9365-9400) was identical in the 10 clones analysed; it was terminated by a UG dinucleotide as described for other isolates. The exact length of the poly(U/UC) region could be determined in all 10 clones and ranged from 72-86 nt. The first 16 nt of the 3′ UTR X region were identical in all clones analysed. The obtained ED43 5′ UTR consensus sequence differed from a published genotype 4a 5′ UTR sequence (D45193) at 1 nt position. For ED43 derived from the infected patient (Y11604) 9, nt 62-340 of the 5′ UTR were determined; this sequence differed from ED43 consensus sequence derived from the chimpanzee plasma pool at 2 nt positions. ED43 consensus ORF sequence, determined in the present study, differed at 125 nt positions (1.4%) and 67 aa positions (2.2%) from Y11604 ORF (FIG. 6).

Differences of at least 2.2% on the aa level were detected in NS2, NS4B, NS5A, and NS5B. Differences of less than 1% were detected in E1 and E2, notably the HVR1 sequence of both isolates was identical at the nt and aa level. At aa 2011 of the ED43 polyprotein, C was found as previously described; C39 in NS5A was described to be critical for replication 62. In contrast, in the infected patient W was reported to be present at this position 9. From 7 other genotype 4a isolates with reported ORF consensus sequence, ED43 consensus sequence differed in 8.8-9.5% at the nt level and at 5.4-6.7% at the aa level (FIG. 6).

Phylogenetic analysis showed that ED43 consensus sequence determined in this study clustering with other genotype 4a isolate sequences, however forming a distinct group with Y11604 (FIG. 1). The 3′ UTR variable region of ED43 consensus sequence determined in the present study was identical to the equivalent sequence of the source patient determined previously and differed at 1 nt from the equivalent sequence of Y11604. Also, high homology was found between 3′ UTR variable region of ED43 and that of several other genotype 4a isolates (FIG. 8). AF009077 had a poly(U/UC) region of 46 nt. The consensus sequence of the first 16 nt of the ED43 X region (nt 9482 to 9497) was identical to the equivalent sequence of AF00907733 and pCV-H77C (Yanagi 1997).

Example 4

Generation of Consensus Clone pED43

The consensus full-length cDNA clone pED43 was constructed in pGEM-9Zf with the following structural elements: (I) 5′ UTR of 340 nt with nt 28-340 being the ED43 nt consensus sequence, while nt 1-27 were derived from D45193. (II) ORF of 9024 nt (nt 341-9364), encoding the ED43 aa consensus sequence. Compared to the ED43 nt consensus sequence, non coding changes are A2458G, A2593G, C3988T, A4459C, C4915T and T5428C; each of these nt changes was present in 1/4 clones analysed. For determination of pED43 nt sequence at nt 1966 and nt 1999, at which no distinct nt consensus was obtained, we used information from 7 clonal sequences previously obtained for this region.

Thus, in pED43 at nt1966, G was chosen, because it was seen in 6/7 of these clones. At nt 1999, C was chosen, seen in 5/7 of these clones. At the other two nt positions without distinct consensus, A was chosen at nt 3751 and C was chosen at nt 3871 in pED43. (III) 3′ UTR of 215 nt (nt 9365-9579) with a variable region of 36 nt (nt 9365-9400) identical to the ED43 nt consensus sequence; with a poly(U/UC) region of 81 nt (nt 9401-9481), chosen from one of the 10 clones analysed; with a conserved X region of 98 nt (nt 9482-9579) determined by the sequence of pCV-H77C (Yanagi 1997), differing at nt position 9556 from X region of the source patient AF009077. An XbaI-site was introduced immediately downstream of the HCV 3′ UTR.

Example 5

RNA Transcripts from pS52 and pED43 do not Lead to Infection of Huh7.5 Hepatoma Cells

Because Huh7.5 cells were shown to be permissive to infection with strain JFH1 and JFH1-based intra- and intergenotypic recombinants including recombinants with Core-NS2 sequence of S52 and ED43, the present inventors tested whether full-length S52 and ED43 RNA transcripts led to productive infection of transfected Huh7.5 cultures.

Thus, replicate cultures were transfected with RNA transcripts from pS52, pED43, and positive control pJ6/JFH1. For J6/JFH1, HCV-Core antigen positive cells were detectable 48 hrs post transfection and viral spread to almost the complete Huh7.5 culture occurred in 4-10 days. In contrast, there were no HCV-Core positive cells detected in cultures transfected with RNA transcripts of pS52 and pED43; these cultures were stained 2 to 3 times per week and followed for 4 weeks. In total four independent transfections with RNA transcripts from pS52; and two transfections with pED43 transcripts were analyzed.

The present inventors further tested whether selected adaptive mutations, leading to efficient growth of intergenotypic recombinants S52/JFH1 (Gottwein 2007) and H77/JFH1 (Yi 2007) as well as JFH1 (Kaul 2007) in hepatoma cell lines, could confer replication capability to the full-length S52. Therefore, we constructed pS52 with single nt exchanges in p7: T2717G (identified in S52/JFH1), in NS3: A4549C (identified in S52/JFH1) or A4097T (identified in H77/JFH1), and in NSSA: G7171C (identified in S52/JFH1) or G7621C (identified in JFH1) (nt positions refer to pS52). Similarly, the present inventors introduced two coding NS2 mutations (A2819G and A3269T), shown to confer cell culture viability to ED43/JFH1 (Scheel 2008), in pED43. However, after transfection of Huh7.5 cells with the respective RNA transcripts, no HCV-Core positive cells were observed; the ED43 (A2819G and A3269T) culture was followed for 1 week, all other cultures were followed for 4 weeks. Thus, cDNA clones pS52 and pED43, with or without putative adaptive mutations, were apparently not replication competent in Huh7.5 cells, and long-term cultures did not lead to adaptation that yielded infectious particles.

Example 6

RNA Transcripts from pS52 are Infectious In Vivo

After intrahepatic transfection of pS52 in vitro RNA transcripts, CH5276 became viremic at week 1 and peak HCV RNA titers of 10⁵-10^(5.5) IU/ml were reached during weeks 6-14 post transfection (FIG. 2A). The ORF sequence of viral genomes recovered at peak HCV titers from serum taken at weeks 7 and 10, respectively, was identical to the sequence of pS52. The animal became anti-HCV positive in a commercial test from week 19 post-infection. However, CH5276 did not develop significant levels of autologous neutralizing antibodies, since pre-incubation of S52/JFH1 viral particles with 1:20 and 1:80 dilutions of week 2 to 32 sera did not lead to >50% of neutralization of S52/JFH1 infectivity in Huh7.5 cells compared to pre-incubation with pre-infection sera (FIG. 2A). CH5276 eventually developed acute hepatitis with elevated serum ALT levels. High ALT levels of ˜100 IU/ml coincided with significant necro-inflammatory liver changes, detected during weeks 19-32. CH5276 became persistently infected with viremia (˜10⁵ IU/ml) at the end of follow-up at week 54. Thus, the constructed S52 consensus sequence was fully functional in vivo.

To further examine the pathogenesis of HCV infection, the present inventors monitored occurrence of HCV specific IFN-γ secreting CD4+/CD8+ T cells in peripheral blood and liver biopsy samples (FIG. 2A). CH5276 peripheral mononuclear cells (PBMC) did not show any IFN-γ secretion above background in ELISpot assays, when stimulated with HCV genotype 3a peptide pools. Intrahepatic IFN-γ secreting CD4+/CD8+ T cells were studied similarly after in vitro expansion and were first detected at week 9 (FIG. 2A). An increase in the percentage of IFN-γ secreting intrahepatic T cells during weeks 11-32 was detected several weeks before occurrence of peak ALT levels and also preceded the most pronounced necro-inflammatory histologic liver changes (observed during weeks 19-32).

Example 7

RNA Transcripts from pED43 are Infectious In Vivo

Immediately after intrahepatic transfection of CH5300 with pED43 in vitro transcripts, the HCV RNA titers increased to peak levels of 10^(4.5)-10^(5.5) IU/ml during weeks 1-8 post transfection (FIG. 2B). The ORF sequence of viral genomes recovered from week 1 and 6 serum did not show any changes compared to the pED43 sequence. CH5300 sero-converted in a commercial test at week 6. However, CH5300 did not develop significant levels of autologous neutralizing antibodies (FIG. 2B). In CH5300, the course of infection was characterized by a fast onset of acute hepatitis with peak serum ALT levels between 100 and 200 IU/ml during weeks 5-10. Peak ALT levels coincided with detection of significant necro-inflammatory liver changes during weeks 5-13. Following week 10, ALT levels decreased to 20 to 70 IU/ml, followed by decrease of liver necro-inflammatory changes. Between week 9 and 54, RNA titers decreased to levels of 10²′⁵-10⁴ IU/ml. CH5300 was persistently infected with viremia (10³-10⁴ IU/ml) at the end of follow-up at week 54. In conclusion, the constructed ED43 consensus sequence was fully functional in vivo.

Intrahepatic CD4+/CD8+ T cells, secreting IFN-γ upon stimulation with HCV genotype 4a peptide pools, were first detected at week 4 post transfection, coinciding with evidence of acute hepatitis. In CH5300, HCV genotype 4a reactive PBMC were detected at time-points, at which strong intrahepatic T cell responses were observed (FIG. 2B).

Discussion

In this study, the present inventors generated and characterized infectious cDNA clones of important genotypes 3a (pS52) and 4a (pED43). Compared to previously developed cDNA clones of genotypes 1a, 1b and 2a as well as consensus sequences of selected isolates of various genotypes and subtypes, pS52 sequence formed a distinct cluster with previously published genotype 3a isolate sequences, while pED43 sequence clustered with described genotype 4a isolate sequences (FIG. 1). Variation between polyprotein consensus sequence of S52, which originated fro Sardinia, Italy, and 3a isolates from New Zealand (NLZ1), Switzerland (452) and Japan (K3a/650) was 3.6-5.9% on the aa level (FIG. 4). Greater variation was observed between sequences of ED43, which originated from Egypt, and several 4a isolates from the Boston area as well as one isolate from Spain (5.4-6.7% on aa level) (FIG. 6). In contrast, ED43 was more similar to genotype 4a isolates obtained from other Egyptian patients. A high degree of variation between S52 and other genotype 3a isolates as well as between ED43 and other genotype 4a isolates was found in genome regions, for which in general a great diversity was described, such as E1, E2 (especially HVR1), p7, NS2, and NS5A (FIGS. 4 and 6).

In contrast, relatively great variation between pED43 and Y11604, which differed in 2.2% of their polyprotein sequence, was also found in NS4B and NS5B, while E1 and E2 were relatively similar (FIG. 6). Interestingly, for ED43 and Y11604, E2 HVR1 was identical on the nt and aa level. ED43 had as Y11604 and other genotype 4a isolates a 4 aa deletion in the interferon sensitivity determining region (ISDR; aa 2210-2245); ED43 and Y11604 ISDR differed at 4 aa (11%) at the N-terminus of this region. Studies of the impact of sequence variations in ISDR on IFN sensitivity will be facilitated by replicon/cell culture systems with genotype specific NS5A (ISDR).

To determine the S52 and ED43 consensus sequence, the inventors studied the quasispecies distribution in standardized acute phase plasma pools. Overall, relatively high genetic heterogeneity was found in genome regions with high genetic diversity such as E1, E2, p7 and NS2 (FIG. 3, 5). In addition, relative high heterogeneity was found in ED43 Core and NS3, two proteins, which in general show less genetic diversity. Another exemption was E2 HVR1, which was identical in all S52 and in all ED43 clones analysed. Genetic heterogeneity in the S52 plasma pool was greater than in the ED43 plasma pool (FIGS. 3 and 5), partly due to occurrence of 2 different quasispecies subpopulations in this pool (Table 1). Different quasispecies subpopulations were previously found in plasma pools of J6 and J4. pED43 cDNA clone reflected the aa consensus sequence, while pS52 had two non consensus aa residues, which were, however, naturally occurring in the S52 pool. As described previously, in pS52 and pED43, the conserved 3″X region was derived from pCV-H77C (Yanagi 1997) but showed close homology to X regions published for other genotype 3a and 4a isolates.

As other previously developed cDNA clones, pS52 and pED43 were not viable in cell culture. Cell culture adaptive mutations identified in JFH1 and JFH1-based intergenotypic recombinants did not lead to cell culture adaptation of pS52 and pED43. The adaptive mechanism of such mutations is not known. They might mediate interaction of HCV proteins derived from different genotypes, however, they might also adapt the respective protein to cell culture, e.g. by facilitation of interaction with cellular binding partners. Proof of functionality of pS52 and pED43 implies proof of functionality of the individual proteins. This knowledge will further development of intergenotypic recombinant cell culture systems containing yet undefined, minimal JFH1 elements, critical for cell culture viability.

Transfection of CH5276 and CH5300 with RNA transcripts of pS52 and pED43 led to robust infection. A course of acute HCV infection, comparable to infection with S52 and ED43, was observed in chimpanzees, which were infected by inoculation with viral particles or intrahepatic transfection with RNA transcripts from various cDNA clones. Even though both animals became persistently infected, significant differences were observed regarding the course of viremia, serum ALT, and cellular immune responses. As previously described for H77 infected chimpanzees, in CH5300 the initial increase in viral RNA (week 1-6) showed a biphasic pattern with a primary rapid and secondary slower slope, separated by a transient decline (week 2) (FIG. 2B).

This decrease in viral replication was suggested to result from activation of innate antiviral defense mechanisms and especially the type-I IFN system, because no intrahepatic HCV reactive T cells but elevated intrahepatic 2′5′ oligoadenylate synthetase 1 mRNA levels were found during the first weeks of HCV infection. Interestingly, for CH5276 the decline in HCV RNA observed at week 2 was far more pronounced and the following increase in HCV RNA more delayed than in CH5300 and acutely infected chimpanzees previously studied (FIG. 2A). In patients, HCV is highly sensitive to treatment with IFN-γ during the acute phase of infection, and in chronically infected individuals genotype 3a is more sensitive to interferon treatment than genotype 1 and 4. Thus, genotype 3a might also be more sensitive to endogenous IFN production during the acute phase of infection. In line with this, higher spontaneous clearance rates have been reported for genotype 3a in one but not other studies. However, even though genotype 2a is supposed to have a relatively great sensitivity to IFN, after transfection of a chimpanzee with RNA transcripts from a genotype 2a cDNA clone, the decline in RNA titers was not as pronounced as for S52. In order to draw conclusions about dependence of early HCV infection kinetics on genotype, more studies with different isolates including monitoring of correlates of innate immunity and other host factors are of importance. During the further course of acute HCV infection different patterns of viremia were observed in various studies. In 5300, a plateau with peak HCV RNA titers (week 5 and 6) was followed by a rapid 2 log decrease of HCV RNA, associated with liver damage most likely mediated by onset of the adaptive immune response (FIG. 2B). This pattern is typically observed in animals that clear HCV but also in some animals that subsequently develop persistent infection; it has not been clarified which immunological and/or viral features are decisive for differential outcomes. In other animals with persistent infection, as observed for CH5276, HCV RNA is consistently detected in serum during the acute phase of infection.

Early, strong, multispecific and sustained CD4+ and CD8+ T cell responses have been associated with viral clearance in humans and chimpanzees. In chimpanzees, occurrence of intrahepatic HCV reactive IFN-γ secreting CD4+ and CD8+ T cells correlated with ALT increase and with at least temporary resolution of viremia. Also occurrence of HCV reactive PBMC, usually present at low frequency, was associated with viral clearance. In general, T cell responses to HCV are delayed; even during a successful adaptive immune response, they occur first after 4-8 weeks post infection. Also, it is frequently seen during HCV infection, that primarily successful looking immune responses, leading to primary control of viremia, all the sudden fail to control infection and viremia rebounds; this might be due to viral escape mechanisms. In both chimpanzees, CH5300, infected with genotype 4a and in CH5276, infected with genotype 3a, we observed intrahepatic T cell responses. In CH5300, T cells occurred early during infection, whereas intrahepatic T cells occurred late in infection in CH5276. In addition, in CH5300, HCV reactive PBMC were present, whereas these were absent in CH5276. Thus, the immune response observed in CH5300 reflected much more a response thought to be efficient against HCV than the immune response seen in CH5276. In line with this, transient decline in viremia was observed for CH5300. In conclusion, infection with S52 and ED43 both triggered an immune response as typically seen in HCV infected chimpanzees and humans underlining the full functionality of the developed cDNA clones pS52 and pED43.

Sequence analysis of viral genomes aimed at demonstrating functionality of the constructed sequences. At the chosen time-points, before onset of adaptive immune responses, S52 and ED43 were genetically stable, indicating full functionality of the developed sequences. This is in contrast to JFH1, which had acquired adaptive mutations already two weeks post transfection.

CH5300 and CH5276 did not develop neutralizing antibodies (ntAB). While ntAB are commonly found in the chronic phase of infection, they are frequently absent during the acute phase. Even though in patients occurrence of nt AB in the acute phase is associated with viral clearance, ntAB are not a pre-requisite for infection control, since they can be absent during resolving infection.

TABLES

TABLE 1

Amino acid positions with genetic heterogeneity of S52 in chimpanzee acute phase plasma pool.

Four overlapping RT-PCR fragments, spanning the complete ORF, and covering (i) aa 1-1019, (ii) aa 1008-1615, (iii) aa 1576-2419, and (iv) aa 2398-3020, were subcloned and analysed. AA Position numbers (AA Pos) refer to pS52. Positions with genetic heterogeneity between clones are shown. Dots represent conserved residues compared to the S52 consensus (S52 Cons) sequence. For non-conserved residues, the aa found at this position is given. Amino acid changes due to defective genomes were not considered to reflect naturally occurring genetic heterogeneity and are therefore not shown: Amino acid frameshift due to nt insertions (nt 6761.1 in clone C8 and nt 9118.1 in clone D17); stop codon (nt change G5618A in clone C19). Grey shadings indicate a minor quasispecies subpopulation, because 2/5 clones of fragment (i), 1/5 clones of fragment (ii), and 2/6 clones of fragment (iii) differed significantly from the majority of clones obtained by subcloning these fragments.

TABLE 2 AA ED43 Pos Cons A1 A81 A41 A55 C3 C5 C2 C4 C1 Core 20 M V • • • 50 R • • Q • 55 R • • • W 79 P • • L • 159 E V • • • 174 F • • • P E1 193 N • S • • 226 C R • • • 265 M • • T • 269 A T • • • 311 G • • • R 333 V • • A • 367 N • • • S 371 V • A • • E2 472 L P • • • 501 S • • F • 529 T N • • • 616 W • • • R 621 T A • • • 658 D • • • V 692 L • • • F p7 756 A • • V • 765 F • • • S 768 A V • • • NS2 826 L • • • P 854 E • K • • 861 I • • • V 921 I • V • • 990 T • S • • 1018 E • • V • NS3 1041 S • G • • 1112 P S • • • 1193 V • A • • 1254 L • • H • 1327 L • P • • 1484 R • • C • 1493 R • K • • 1519 E • • • G 1526 A • • V • 1551 C • • R • 1554 H R • • • 1566 T • • • A 1577 K • • E • 1583 F • • S • NS4A 1675 L • • • P 1688 L • R • • NS4B 1713 K E • • • • • • • • 1791 M • T • • • 1885 A • T • • • 1905 H • • R • • 1954 T • • P • • NS5A 2088 E • • • • G 2130 L • • • • I 2369 T • I • • • NS5B 2455 Y H • • • • 2461 S P • • • • 2547 N • • • D • 2571 R • • W • • 2582 L • • • • P 2627 S • • • • P 2750 N • • S • • 2806 E G • • • • 2884 H Q • • • • 2935 A • • • • V

Amino acid positions with genetic heterogeneity of ED43 in chimpanzee acute phase plasma pool.

Two overlapping RT-PCR fragments, covering (i) aa 1-1763, and (ii) aa 1713-3008, were subcloned and analysed. AA Position numbers (AA Pos) refer to pED43. Positions with genetic heterogeneity between clones are indicated. Dots represent conserved residues compared to the ED43 consensus (ED43 cons) sequence. For non-conserved residues, the aa found at this position is given. A stop codon (resulting from nt change at position 1930 in clone A41) was not considered to reflect naturally occurring genetic heterogeneity and is not shown.

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The invention claimed is:
 1. An isolated nucleic acid molecule comprising a consensus sequence construction which encodes a human hepatitis C virus of genotype 3, wherein said molecule is capable of infectivity in vivo, and wherein said molecule encoding human hepatitis C virus of genotype 3a encodes the constructed amino acid sequence according to SEQ ID NO: 1 or a constructed amino acid sequence that has a sequence identity of at least 98% over the entire length of SEQ ID NO:
 1. 2. A recombinant DNA construct comprising a nucleic acid molecule according to claim
 1. 3. An RNA transcript of the DNA construct according to claim
 2. 4. A cell transfected with the DNA construct of claim
 2. 5. A cell transfected with the RNA transcript according to claim
 3. 6. A hepatitis C virus produced by the cell according to claim
 4. 7. A hepatitis C virus produced by the cell according to claim
 5. 8. A hepatitis C virus whose genome comprises a nucleic acid molecule according to claim
 1. 9. A method for producing a hepatitis C virus comprising transfecting a host cell with the RNA transcript according to claim
 3. 10. A method for assaying candidate antiviral agents for activity against HCV, comprising: a) exposing a cell containing the hepatitis C virus according to claim 8 to the candidate antiviral agent; and b) measuring the presence or absence of hepatitis C virus replication or correlates thereof in the cell of step (a).
 11. A method for determining the susceptibility of cells in vitro to support HCV infection, comprising the steps of: a) growing animal cells in vitro; b) transfecting into said cells the nucleic acid according to claim 1; and c) determining if said cells show indicia of HCV replication.
 12. A composition comprising a nucleic acid molecule according to claim 1 suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.
 13. The nucleic acid molecule according to claim 1, wherein said molecule encoding human hepatitis C virus of genotype 3a comprises a nucleic acid sequence having a sequence identity of at least 98% over the entire length of SEQ ID NO:
 3. 14. The nucleic acid molecule according to claim 1, wherein said molecule encoding human hepatitis C virus of genotype 3a comprises a nucleic acid sequence having the sequence of SEQ ID NO:
 3. 